Prostaglandins (PGs) are clinically important lipid signaling molecules derived from membrane phospholipids. They have been implicated in a variety of developmental, physiological, and pathophysiological processes, including fertilization, inflammation, metabolic disease, Alzheimer's disease, and cancer. PG isomers called isoprostanes are used as markers of oxidative stress. The current dogma is that PG-endoperoxide synthase (i.e. Cox) enzymes are the sole enzymes capable of initiating PG synthesis. However, this model had not been directly tested. Cox enzymes are targets of NSAIDs, widely used drugs for treating pain and inflammation. This application is driven by the unexpected discovery that C. elegans and mice synthesize specific PGF2? stereoisomers independent of Cox. C. elegans PGs have a critical role in fertilization and are dynamically regulated by pheromones and nutritional cues. The objective of this proposal is to delineate the Cox- independent PG biosynthesis mechanism. The central hypothesis is that an evolutionarily conserved Cox- independent metabolic pathway synthesizes PGF1? and PGF2? stereoisomers in a tissue-specific fashion. Three independent aims are proposed to test the central hypothesis. Aim 1 will identify genes essential for Cox-independent PG synthesis. The simple roundworm model C. elegans will be used for these studies because a genetic screening method has been developed to identify these genes. Liquid chromatography tandem mass spectrometry (LC-MS/MS) will determine PG levels in mutant worms. Gene replacement experiments will assess functional conservation of mouse homologs. Aim 2 will identify biochemical steps in C. elegans PG metabolism. Stable isotope labeling and mass spectrometry methods will identify PG metabolites. Nuclear magnetic resonance will determine stereostructure of major worm PGs. Aim 3 will investigate Cox- independent PG metabolism in mice. LC-MS/MS will identify PGs in Cox null mouse pups and isolated tissues. Pharmacologic studies in Cox null embryonic fibroblast cultures will test pathway sensitivity to NSAIDs. A long- term goal is develop the mouse as a model to study Cox-independent PGs. These studies will help define a second widely used pathway for PG synthesis and provide a roadmap for delineating this pathway in humans. The results will provide genetic tools for discovering new PG functions in development and disease. By transforming PG biochemistry, new avenues will emerge for developing therapeutics to treat cancer, glaucoma, cardiovascular diseases, and other human disorders.